SUMMARY Platinating agents such as cis-platinum (cisplatin, CDDP) are important genotoxic (DNA-damaging) drugs used for the treatment of many cancers. Unfortunately, there are major limitations to the successful treatment of cancer with platinating agents including the emergence of drug-resistant tumor cells and toxic side effects. It is crucial to devise better means of targeting cisplatin-resistant cancer cells and reducing cisplatin toxicity in order to attain the full therapeutic potential of platinum drugs. Trans-Lesion Synthesis (TLS) is a major mechanism by which cancer cells acquire tolerance to DNA damage from platinating agents. Therefore, the long-term goal of this project is to inhibit TLS in cancer patients, thereby improving our treatment of tumors that resist conventional chemotherapies. The objective in this application is to develop small molecules that inhibit an interaction between the TLS proteins DNA Polymerase eta (Polh) and PCNA that is crucial for DNA damage tolerance. The central hypothesis is that therapeutic inhibition of Polh-PCNA interactions will sensitize cancer cells to killing by platinating agents and lower the therapeutic doses of cisplatin. The rationale is that combination therapies comprising TLS inhibitors and platinating agents will provide a powerful strategy for ameliorating chemoresistance and toxic side effects of high-dose cisplatin therapy. Based on strong preliminary studies, our hypothesis will be tested using three Specific Aims (SAs): (1) Develop and Validate a High-Throughput Screen (HTS) for inhibitors of the Polh-PCNA interaction. (2) Develop and Validate Secondary Assays for specific inhibition of the Polh-PCNA interaction. (3) Characterize the effects of active compounds on cisplatin-sensitivity of cancer cell lines. We have successfully reconstituted the Polh-PCNA interaction in vitro and in SA1 this biochemical assay will be optimized and adapted for HTS. We will perform a pilot screen using a commercially-available small (1280) Library of Pharmacologically Active Compounds and 5000 randomly-selected compounds from the UNC diversity library. In SA2 candidate inhibitors identified by the pilot screen will be confirmed and retested in potency curves, then subject to a panel of independent secondary assays to eliminate false positives. In SA3, we will determine the effects of inhibitors we identify on viability and CDDP-tolerance of a panel of human cancer cell lines and primary human cells. Following completion of the proposed studies and validation of our assays we will be poised to conduct a HTS using large (>200,000) diversity libraries. The proposed work will target a regulated interaction between Polh and the mono-ubiquitinated form of PCNA. Thus, identification of compounds that inhibit interactions involving mono- ubiquitinated proteins would provide a new paradigm for therapeutic strategies involving ubiquitin signaling and would undoubtedly facilitate future drug discovery efforts. Therefore the proposed strategy is highly innovative. The proposed research is significant because it aims to reduce the problem of mortality due to cancer in the United States.